Biochemical reaction temperature measuring device and method based on solid-state spin sensor

Abstract

The application provides a biochemical reaction temperature measuring device and method based on a solid-state spin sensor. The device comprises: a sample module, which is prearranged with a sample to be measured and solid-state spin nanoparticles as temperature probes, and is used for completing biochemical reactions of the sample to be measured and measuring temperature changes in the biochemical reactions; a temperature control module, which is used for keeping the environmental temperature and humidity of the area occupied by the sample module stable; a pump light module, which is used for focusing an excitation light beam to the solid-state spin nanoparticles, so that target solid-state spin nanoparticles in the light beam range of the excitation light beam emit fluorescence; a microwave module, which is used for radiating a microwave field to the solid-state spin nanoparticles; a collection module, which is used for processing fluorescence light beams corresponding to the fluorescence to obtain fluorescence signals; and a processing module, which is used for converting the fluorescence signals into characteristic signals, and determining a temperature change measurement result of the biochemical reaction process according to the characteristic signals.

Description

Technical Field

[0001] This invention relates to the field of nanocalorimetry, and more specifically, to a device and method for measuring the temperature of biochemical reactions based on a solid-state spin sensor. Background Technology

[0002] The generation and absorption of heat are ubiquitous phenomena in biochemical activities. Temperature monitoring can provide important information about processes such as biomolecular interactions, conformational changes, and enzyme kinetics.

[0003] Temperature measurement methods such as isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC) have been widely used in many scientific discoveries due to their advantages of being label-free and requiring no analyte fixation. However, these measurements typically require large sample volumes, usually hundreds of microliters, which hinders their application in high-throughput detection and low-sample-consumption scenarios, such as drug screening and catalytic reaction optimization.

[0004] With recent advancements in microfluidic technology for manipulating micron and nanodroplets, the demand for label-free temperature measurements of correspondingly small volumes is growing, making the development of nanoscale thermometers with high-temperature sensitivity and long-term chemical stability a challenge. Isoelectric thermometers, such as thermistors and thermopile thermometers, typically range in size from tens to hundreds of μm, requiring a 2-5 μm thick protective layer between the thermometer and the reaction liquid. Furthermore, while fluorescence-based thermoresponsive molecules can achieve nanoscale applications, most measurement methods suffer from signal instability due to the influence of local pH, ion concentration, and refractive index. Observations of submicron-scale biochemical reactions with resolutions better than 100 mK are also lacking. Summary of the Invention

[0005] In view of this, the present invention provides a biochemical reaction temperature measurement device and method based on a solid-state spin sensor.

[0006] One aspect of the present invention provides a biochemical reaction temperature measurement device based on a solid-state spin sensor, comprising: a sample module pre-loaded with a sample to be tested and solid-state spin nanoparticles serving as temperature probes, wherein the sample module is used to perform the biochemical reaction of the sample to be tested, and the solid-state spin nanoparticles are used to measure the temperature change during the biochemical reaction process; a temperature control module for controlling the ambient temperature and humidity of the area occupied by the sample module to remain stable; a pump light module for generating an excitation beam and focusing the excitation beam onto the solid-state spin nanoparticles so that the target solid-state spin nanoparticles within the beam range of the excitation beam emit fluorescence; a microwave module for generating a microwave field and radiating the microwave field onto the solid-state spin nanoparticles; a collection module for collecting a fluorescence beam corresponding to the fluorescence and processing the fluorescence beam to obtain a fluorescence signal; and a processing module for receiving the fluorescence signal, converting the fluorescence signal into a feature signal, and determining the temperature change measurement result of the biochemical reaction process based on the feature signal.

[0007] Optionally, the solid spin nanoparticles include at least one of the following: nanoparticles containing a temperature-sensitive spin sensor, and composite probes combining a temperature-responsive material and the aforementioned spin sensor.

[0008] Optionally, the sample to be tested includes a first sample and a second sample. The sample module includes: a microdroplet controller for generating a predetermined volume of the first sample; a reaction chamber for loading the second sample and the solid spin nanoparticles, and providing reaction space for the biochemical reaction process of the first sample and the second sample; a microfluidic tube for introducing the first sample generated by the microdroplet controller into the reaction chamber; and a displacement module for supporting the reaction chamber and controlling the solid spin nanoparticles loaded in the reaction chamber to be locked at the focusing point of the microscope objective.

[0009] Optionally, the microwave module includes: a microwave source for generating a microwave field with modulated spin states; a microwave power amplifier for enhancing the power of the microwave field; and a radiation component for radiating the microwave field to the solid-state spin nanoparticles.

[0010] Optionally, the collection module includes: a filter for filtering the fluorescence beam; an achromatic lens for focusing the fluorescence beam onto a pinhole; the pinhole for spatial filtering of the fluorescence beam; a beam splitter for splitting the filtered and spatially filtered fluorescence beam and sending it to a spectrometer and an avalanche diode; the avalanche diode for converting the fluorescence beam into fluorescence counting for recording the number of fluorescence photons; and the spectrometer for converting the fluorescence beam into a spectral signal.

[0011] Optionally, the pump light module includes: a light source for generating an excitation beam with a wavelength corresponding to the aforementioned spin sensor; a modulator for modulating the intensity of the excitation beam; an fiber collimator for projecting the modulated excitation beam onto a dichroic mirror; the dichroic mirror for transmitting the excitation beam to a microscope objective and transmitting the fluorescence beam to the aforementioned collection module; and the microscope objective for focusing the excitation beam onto the aforementioned solid spin nanoparticles and collecting the fluorescence beam.

[0012] Optionally, the temperature control module includes: an outer temperature control submodule, configured to surround the microscope objective and the sample module, for providing a stable ambient temperature for the microscope objective and the sample module; and an inner temperature control submodule, configured to surround the sample module, for providing a stable ambient temperature and humidity for the solid spin nanoparticles.

[0013] Optionally, the inner temperature control submodule includes: a surrounding heating plate, configured to surround the reaction chamber in conjunction with the displacement module, for providing a stable ambient temperature for the solid spin nanoparticles; a water tank, disposed around the surrounding heating plate, for providing a stable ambient humidity for the solid spin nanoparticles; a reference thermometer, configured to be connected to the reaction chamber, for measuring the ambient temperature of the reaction chamber; and a temperature controller, configured to be connected to the reference thermometer, for controlling the power of the surrounding heating plate according to the ambient temperature measured by the reference thermometer.

[0014] Another aspect of the present invention provides a method for measuring the temperature of a biochemical reaction based on a solid-state spin sensor, implemented based on the aforementioned biochemical reaction temperature measurement device based on a solid-state spin sensor. The method includes: using a temperature control module to maintain a stable ambient temperature and humidity in a sample module, wherein the sample module includes solid-state spin nanoparticles serving as temperature probes and a sample to be tested for a biochemical reaction; using a pump light module to apply an excitation beam to the solid-state spin nanoparticles placed in the sample module; using a microwave module to apply a microwave field to the solid-state spin nanoparticles placed in the sample module; using an avalanche diode to acquire a fluorescence count of the fluorescent photons emitted by the solid-state spin nanoparticles; using a microwave source to acquire the microwave frequency of the microwave field; using a processing module to control the simultaneous application of the excitation beam and the microwave field, and recording the fluorescence count; when a change in the fluorescence count is detected, acquiring a target microwave frequency matching the spin energy level of the solid-state spin nanoparticles; and determining the temperature change measurement result of the biochemical reaction process based on the target microwave frequency.

[0015] Another aspect of the present invention provides a method for measuring the temperature of a biochemical reaction based on a solid-state spin sensor, implemented based on the aforementioned biochemical reaction temperature measurement device based on a solid-state spin sensor. The method includes: using a temperature control module to maintain a stable ambient temperature and humidity within a sample module, wherein the sample module includes solid-state spin nanoparticles serving as temperature probes and a sample to be tested for a biochemical reaction; using a pump light module to apply an excitation beam to the solid-state spin nanoparticles placed within the sample module; using a spectrometer to acquire spectral information of the fluorescence emitted by the solid-state spin nanoparticles; and using a processing module to determine the temperature change measurement result of the biochemical reaction process based on the spectral information.

[0016] According to embodiments of the present invention, a technique is employed that utilizes a sample module pre-placed with a test sample and solid-state spin nanoparticles as temperature probes to complete the biochemical reaction of the test sample. Combined with processing by a temperature control module, a pump light module, and a microwave module, the temperature change measurement results of the biochemical reaction process are obtained based on the collected characteristic signals. Because the solid-state spin nanoparticles used have high stability to the biochemical environment, they are suitable for measuring various complex biochemical reaction processes. Using nanoscale particles as probes achieves sub-micron spatial resolution, and combined with the test sample, the measurement volume can be reduced to the order of liters. Simultaneously, the solid-state spin nanoparticles exhibit high temperature sensitivity, giving the temperature measurement device advantages such as high chemical stability, high temperature sensitivity, and low sample consumption. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 A schematic diagram illustrates the structure of a biochemical reaction temperature measurement device based on a solid-state spin sensor according to an embodiment of the present invention.

[0019] Figure 2 A schematic diagram of a sample module in a non-isothermal calorimetric mode according to an embodiment of the present invention is shown.

[0020] Figure 3 A schematic diagram of a sample module in isothermal calorimetry mode according to an embodiment of the present invention is illustrated.

[0021] Figure 4 A schematic diagram of the inner temperature control submodule according to an embodiment of the present invention is shown;

[0022] Figure 5 The optical path diagram of a biochemical reaction temperature measurement device based on a solid-state spin sensor according to an embodiment of the present invention is illustrated schematically.

[0023] Figure 6 A flowchart illustrating a method for measuring the temperature of a biochemical reaction based on a solid-state spin sensor, implemented using the ODMR method according to an embodiment of the present invention, is shown schematically.

[0024] Figure 7A A schematic diagram illustrating the counting principle of a single fluorescence count according to an embodiment of the present invention is shown.

[0025] Figure 7B A schematic diagram illustrating the determination of the resonant frequency based on microwave frequency and fluorescence count according to an embodiment of the present invention is shown.

[0026] Figure 7C A schematic diagram illustrating the determination of temperature change based on resonant frequency according to an embodiment of the present invention is shown.

[0027] Figure 8 A flowchart illustrating a method for measuring the temperature of a biochemical reaction based on a solid-state spin sensor, implemented using an all-optical method according to an embodiment of the present invention, is shown.

[0028] Figure 9 The schematic diagram illustrates the principle of temperature measurement using the All-optical method according to an embodiment of the present invention;

[0029] Figure 10 An example diagram illustrating the temperature response coefficient measurement using a spectroscopic method according to an embodiment of the present invention is shown.

[0030] Figure 11A This diagram illustrates the detection of temperature changes during an acid-base neutralization reaction using an all-optical method according to an embodiment of the present invention.

[0031] Figure 11B This illustration schematically shows the detection results of the stability of acid-base neutralization reactions using an all-optical method according to an embodiment of the present invention; and

[0032] Figure 12 The diagram illustrates the detection of temperature changes during the ATP enzymatic hydrolysis process using an all-optical method according to an embodiment of the present invention. Detailed Implementation

[0033] Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. However, it should be understood that these descriptions are exemplary only and are not intended to limit the scope of the invention. In the following detailed description, numerous specific details are set forth to provide a thorough understanding of the embodiments of the invention for ease of explanation. However, it will be apparent that one or more embodiments may be practiced without these specific details. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concept of the invention.

[0034] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. The terms “comprising,” “including,” etc., as used herein indicate the presence of the stated features, steps, operations, and / or components, but do not exclude the presence or addition of one or more other features, steps, operations, or components.

[0035] All terms used herein (including technical and scientific terms) have the meanings commonly understood by those skilled in the art, unless otherwise defined. It should be noted that the terms used herein are to be interpreted in a manner consistent with the context of this specification, and not in an idealized or overly rigid way.

[0036] When using expressions such as "at least one of A, B, and C," the expression should generally be interpreted in accordance with the meaning commonly understood by a person skilled in the art (e.g., "a system having at least one of A, B, and C" should include, but is not limited to, systems having A alone, having B alone, having C alone, having A and B, having A and C, having B and C, and / or having A, B, and C, etc.). Similarly, when using expressions such as "at least one of A, B, or C," the expression should generally be interpreted in accordance with the meaning commonly understood by a person skilled in the art (e.g., "a system having at least one of A, B, or C" should include, but is not limited to, systems having A alone, having B alone, having C alone, having A and B, having A and C, having B and C, and / or having A, B, and C, etc.).

[0037] One embodiment of the present invention provides a biochemical reaction temperature measurement device based on a solid-state spin sensor.

[0038] Figure 1 A schematic diagram of a biochemical reaction temperature measurement device based on a solid-state spin sensor according to an embodiment of the present invention is shown.

[0039] like Figure 1 As shown, the biochemical reaction temperature measurement device based on a solid-state spin sensor may include a pump light module 1, a sample module 2, a temperature control module 3, a microwave module 4, a collection module 5, and a processing module 6.

[0040] Pump light module 1 is used to generate excitation beam 10 and focus excitation beam 10 onto solid spin nanoparticles pre-placed in sample module 2, so that the target solid spin nanoparticles within the beam range of excitation beam 10 emit fluorescence 20.

[0041] Sample module 2 is pre-loaded with the sample to be tested and solid spin nanoparticles as temperature probes. Sample module 2 can be used to complete the biochemical reaction of the sample to be tested, and the solid spin nanoparticles can be used to measure the temperature change during the biochemical reaction process.

[0042] Temperature control module 3 is used to control the ambient temperature and humidity of the area occupied by sample module 2 to maintain stability.

[0043] Microwave module 4 is used to generate microwave field 40 and radiate microwave field 40 onto solid-state spin nanoparticles. The microwave field can be used to modulate the spin state of solid-state spin nanoparticles.

[0044] Collection module 5 is used to collect the fluorescence beam corresponding to fluorescence 20, and to process the fluorescence beam to obtain fluorescence signal 50.

[0045] The processing module 6 is used to receive the fluorescence signal 50, convert the fluorescence signal 50 into a characteristic signal, and determine the temperature change measurement result of the biochemical reaction process based on the characteristic signal.

[0046] According to an embodiment of the present invention, the characteristic signal may include a microwave frequency signal. When using optically probed magnetic resonance (ODMR) thermometry, the processing module 6 can control the pump light module 1 and the microwave module 4 to operate simultaneously, applying both the excitation beam and the microwave field to the solid spin nanoparticles in the sample module 2. In this case, the target microwave frequency 30, where the microwave field 40 generated by the microwave module 4 matches the spin energy level of the spin sensor in the solid spin nanoparticles, can be used as the characteristic signal processed by the processing module 6.

[0047] According to another embodiment of the present invention, the feature signal may further include a spectral signal. When using an all-optical temperature measurement method, the microwave module 4 may not be included in the device. In this case, the spectral signal corresponding to the fluorescence signal can be collected by the collection module 5, and the spectral signal can be used as a feature signal processed by the processing module 6.

[0048] According to an embodiment of the present invention, after determining the characteristic signal, the temperature change around the solid spin nanoparticle can be measured based on the characteristic signal, thereby obtaining the temperature change measurement results during the biochemical reaction process.

[0049] According to embodiments of the present invention, the sample to be tested may include various reaction solutions or reaction samples capable of undergoing biochemical reactions. In cases where it is necessary to measure temperature changes during a biochemical reaction using solid spin nanoparticles, the sample to be tested can be immersed in solid spin nanoparticles.

[0050] The following describes specific embodiments in conjunction with the accompanying drawings. Figure 1 The biochemical reaction temperature measurement device based on a solid-state spin sensor shown is described in further detail.

[0051] According to embodiments of the present invention, solid-state spin nanoparticles include at least one of the following: nanoparticles containing a temperature-sensitive spin sensor, and composite probes combining a temperature-responsive material and the spin sensor.

[0052] According to embodiments of the present invention, solid-state spin nanoparticles can contain spin sensors. For example, solid-state spin nanoparticles can be selected from spin sensors such as NV (Nitrogen-Vacancy) centers, SiV centers, and GeV centers. Spin sensors, represented by NV centers, are an emerging type of quantum thermal probe. They can exhibit high sensitivity at the nanoscale, as well as extremely high chemical stability and thermal conductivity. They can directly contact reaction solutions and adapt to various complex chemical environments, which is beneficial for detecting temperature changes in various biochemical reactions.

[0053] According to embodiments of the present invention, nanoparticles can also be combined with other temperature-responsive materials to form composite probes. Temperature-responsive materials may include, for example, copper-nickel materials, and are not limited thereto.

[0054] According to embodiments of the present invention, the size and implantation concentration of the solid spin nanoparticles can be selected as needed. For example, the size of the solid spin nanoparticles can be selected as 100 nm, and the implantation concentration can be selected as 3.5 ppm.

[0055] According to an embodiment of the present invention, the pump light module may include: a light source for generating an excitation beam with a wavelength corresponding to the spin sensor; and a microscope objective for focusing the excitation beam onto the solid spin nanoparticles and collecting the fluorescence beam emitted by the solid spin nanoparticles.

[0056] According to embodiments of the present invention, the light source may include a laser light source, or other light sources. The laser light source may include a semiconductor laser, or other types of lasers. Given the advantages of laser light sources such as high collimation, high brightness, and good monochromaticity, a laser light source can be selected to achieve better results. The emission wavelength of the light source can be determined based on the excitation wavelength corresponding to the solid-state spin nanoparticles. For example, corresponding to the NV color center spin sensor, a wavelength of 532 nm or 593 nm can be selected.

[0057] According to embodiments of the present invention, the pump light module may further include at least one of the following: an optical fiber coupler for coupling an excitation beam emitted by a light source into an optical fiber; a modulator for modulating the intensity of the excitation beam; an optical fiber collimator for projecting the modulated excitation beam onto a dichroic mirror; and a dichroic mirror for transmitting the excitation beam to a microscope objective and transmitting a fluorescence beam emitted by solid-state spin nanoparticles to a collection module.

[0058] According to embodiments of the present invention, the modulator can be any one of an acousto-optic modulator, an optical fiber coupled acousto-optic modulator, etc. When the modulator is an optical fiber coupled acousto-optic modulator, the optical fiber coupler can be connected to the optical fiber coupled acousto-optic modulator via an optical fiber, and the optical fiber collimator can be connected to the optical fiber coupled acousto-optic modulator via an optical fiber. The modulator can receive TTL signals from the processing module. The TTL signals can be generated by the PCI (Peripheral Component Interconnect) board of the processing module and can be controlled by a program.

[0059] According to embodiments of the present invention, the sample to be tested may include a first sample and a second sample. The sample module may include: a microdroplet controller for generating a predetermined volume of the first sample; a reaction chamber for loading the second sample and solid spin nanoparticles, and providing reaction space for the biochemical reaction process of the first and second samples; a microfluidic tube for introducing the first sample generated by the microdroplet controller into the reaction chamber; and a displacement module for supporting the reaction chamber and controlling the solid spin nanoparticles loaded within the reaction chamber to lock at the focusing point of the microscope objective.

[0060] According to embodiments of the present invention, the microdroplet controller can control the volume of the generated reaction liquid. For example, a first sample in the form of microdroplets can be generated, with a volume controllable to 1 μL. The first sample can be introduced into the reaction chamber through a microfluidic tube to undergo a biochemical reaction with a second sample loaded in the reaction chamber. The second sample loaded in the reaction chamber can immerse solid spin nanoparticles, which can detect the temperature change in the reaction chamber from the start of the biochemical reaction to its completion in real time.

[0061] According to embodiments of the present invention, the reaction chamber can be composed of a PMMA (Polymethyl methacrylate, plexiglass) ring and a heat insulation layer to form a heat-insulated microdroplet reaction chamber. The heat insulation layer can be composed of double-layered glass with a certain gap. An air insulation layer or a vacuum insulation layer can be selected to insulate against the environment as needed. Solid spin nanoparticles can be fixed on a glass substrate above the heat insulation layer. It should be noted that other micro / nano structures can also be used to design the reaction chamber.

[0062] According to embodiments of the present invention, the displacement module may include a coarse-adjustment three-dimensional displacement stage with lower precision and a scanning three-dimensional displacement stage with higher precision, which can be used to achieve large-range movement and precise positioning. By mounting the reaction chamber on the displacement module and adjusting the displacement device, the solid spin nanoparticles fixed on the heat insulation layer of the reaction chamber can be locked to the focusing point of the microscope objective.

[0063] Figure 2 A schematic diagram of a sample module in a non-isothermal calorimetric mode according to an embodiment of the present invention is shown.

[0064] like Figure 2 As shown, 26 is a heat insulation layer used to reduce heat dissipation within the reaction chamber. 24 is a PMMA ring; the combination of the heat insulation layer 26 and the PMMA ring 24 forms the reaction chamber 25. 22 is a petri dish, which can be used to construct a smaller external space for the reaction chamber 25. The reaction chamber 25 can provide a mixing and reaction area for the first and second samples performing the biochemical reaction. 27 is a solid spin nanoparticle, which can be fixed on the glass substrate above the heat insulation layer 26, i.e., fixed at the bottom of the reaction chamber 25. 23 is a microfluidic tube, and 28 is a microdroplet controller; their combination, for example, can drop a predetermined volume of the first sample into the reaction chamber 25, where it reacts with the second sample pre-loaded with the solid spin nanoparticles 27 immersed in the reaction chamber 25. During the biochemical reaction, the solid spin nanoparticles 27 can detect temperature changes in the relevant biochemical reaction process while maintaining a stable sample module temperature.

[0065] According to embodiments of the present invention, under the condition of maintaining a stable inner ambient temperature, the heat change of the biochemical reaction can also be directly measured using isothermal calorimetry. That is, by using heat-absorbing materials, the heat released by the biochemical reaction is absorbed, so that the thermal equilibrium temperature inside the reaction chamber is always maintained at the same temperature as the ambient temperature.

[0066] Figure 3 A schematic diagram of a sample module in isothermal calorimetry mode according to an embodiment of the present invention is illustrated.

[0067] like Figure 3As shown, taking an exothermic biochemical reaction as an example, 291 is a photocooling material that can be placed inside the reaction chamber 25 as an endothermic material in the isothermal calorimetric mode. The photocooling material 291 can adjust the power of the cooling light 292 to perform corresponding endothermic operations based on the exothermic state of the biochemical reaction in the reaction chamber 25, so that the thermal equilibrium temperature of the reaction chamber 25 is consistent with the ambient temperature, i.e., the temperature change inside the reaction chamber is zero. Here, the solid spin nanoparticles 27 are used to monitor the temperature change inside the reaction chamber and feed it back to the controller of the cooling light 292, facilitating the adjustment of its power to achieve the goal of making the thermal equilibrium temperature of the reaction chamber 25 consistent with the ambient temperature. During the experiment, the power change of the cooling light 292 is recorded. Combined with the calibration of the endothermic power of the photocooling material 291, the exothermic heat of the biochemical reaction occurring in the reaction chamber 25 can be determined, and the temperature change measurement results of the relevant biochemical reaction can be obtained. The photocooling material 291 and the solid spin nanoparticles 27 placed inside the reaction chamber can be isolated from each other in a manner with good thermal contact.

[0068] According to embodiments of the present invention, the temperature control module may include an outer temperature control submodule and an inner temperature control submodule. The outer temperature control submodule may be configured to surround the microscope objective and the sample module, and can be used to provide a stable ambient temperature for the microscope objective and the sample module. The inner temperature control submodule may be configured to surround the sample module, or it may be configured to surround the reaction chamber of the sample module, and can be used to provide a stable ambient temperature and humidity for the solid spin nanoparticles.

[0069] According to an embodiment of the present invention, the inner temperature control submodule may include: a surrounding heating plate, configured to surround the reaction chamber in conjunction with a displacement module, for providing a stable ambient temperature for the solid spin nanoparticles; a water tank, disposed around the surrounding heating plate, for providing a stable ambient humidity for the solid spin nanoparticles; a reference thermometer, configured to be connected to the reaction chamber, for measuring the ambient temperature of the reaction chamber; and a temperature controller, configured to be connected to the reference thermometer, for controlling the power of the surrounding heating plate according to the ambient temperature measured by the reference thermometer.

[0070] Figure 4 A schematic diagram of the inner temperature control submodule according to an embodiment of the present invention is shown.

[0071] like Figure 4As shown, 324 is a surrounding heating plate, which can be used to provide a stable local temperature for the reaction chamber 25 and the sample and solid spin nanoparticles 27 inside. 323 is a water tank, which can be used to provide stable local humidity for the reaction chamber 25 and the sample and solid spin nanoparticles 27 inside, reducing the evaporation of the reaction liquid and sample. 322 is a reference thermometer, which can detect the ambient temperature around the reaction chamber in real time. 321 is a temperature controller, which can display the temperature value detected by the reference thermometer 322 in real time, and can adjust the heating power of the surrounding heating plate 324 according to the temperature value to maintain the stability of the ambient temperature around the reaction chamber. Combining the reference thermometer 322 and the temperature controller 321, a stable ambient temperature can be provided for the biochemical reaction process of the sample.

[0072] According to an embodiment of the present invention, the microwave module 4 may include: a microwave source for generating a microwave field modulated with a spin state; a microwave power amplifier for enhancing the power of the microwave field; and a radiation assembly for radiating the microwave field to solid-state spin nanoparticles.

[0073] According to embodiments of the present invention, the microwave field may include an electromagnetic magnetic field. A radiation component may be installed near the sample module and can be used to radiate the microwave field generated by the microwave source onto the solid-state spin nanoparticles to modulate their spin state. The resulting microwave module can emit electromagnetic waves to the solid-state spin nanoparticles, which can modulate the spin state of the spin sensors within the solid-state spin nanoparticles and the spin sensors within the composite probe.

[0074] According to embodiments of the present invention, the collection module may include: an avalanche diode for converting the fluorescence beam emitted by the solid-state spin nanoparticles into a fluorescence count for recording the number of fluorescence photons, and outputting the fluorescence count to the processing module; and a spectrometer for converting the fluorescence beam emitted by the solid-state spin nanoparticles into a spectral signal, and outputting the spectral signal to the processing module.

[0075] According to an embodiment of the present invention, the avalanche diode can operate in a mode using the ODMR method for temperature measurement. In this mode, the microwave module can be deployed and operated simultaneously. The spectrometer can operate in a mode using the all-optical method for temperature measurement. In this mode, the relevant functions of the microwave module can be disabled.

[0076] According to an embodiment of the present invention, the collection module 5 may further include at least one of the following: a filter for filtering the fluorescence beam and removing interference signals; an achromatic lens for focusing the fluorescence beam onto a pinhole; a pinhole for spatial filtering of the fluorescence beam, the size of which can be selected according to the required spatial resolution, for example, a pinhole with a diameter of 50 μm; and a beam splitter for splitting the filtered and spatially filtered fluorescence beam and sending it to the spectrometer and avalanche diode.

[0077] According to embodiments of the present invention, the processing module may include a computer-controlled processing module. The computer-controlled processing module can process the collected fluorescence signals, converting them into characteristic signals such as target microwave frequencies and spectral signals, and can obtain temperature change measurement results of related biochemical reaction processes through changes in these characteristic signals.

[0078] Figure 5 The optical path diagram of a biochemical reaction temperature measurement device based on a solid-state spin sensor according to an embodiment of the present invention is shown schematically.

[0079] like Figure 5 As shown, the pump light module 1 and the collection module 5 can constitute a confocal microscopy device. This microscopy device may include a light source 11, a light intensity attenuator 18, a first reflecting mirror 19, an optical fiber coupler 14, a modulator 15, an optical fiber collimator 16, a dichroic mirror 12, a second reflecting mirror 17, a microscope objective lens 13, a filter 55, a first achromatic lens 56, a pinhole 54, a second achromatic lens 57, a beam splitter 53, a third achromatic lens 58, an avalanche diode 51, and a spectrometer 52, etc.

[0080] According to an embodiment of the present invention, see Figure 5 As shown, the excitation beam can be emitted from the light source 11, pass through the attenuator 18 and the first reflecting mirror 19, and then be coupled into the fiber coupler 14. It is then connected to the modulator 15 through an optical fiber, and finally exits through the fiber collimator 16, becoming free light. The free light is reflected by the dichroic mirror 12 and the second reflecting mirror 17 and enters the microscope objective 13, where it can be focused onto the solid spin nanoparticles 27, causing the solid spin sensor within the beam range to emit fluorescence.

[0081] According to an embodiment of the present invention, see Figure 5As shown, when solid-state spin nanoparticles emit fluorescence under excitation light, the fluorescence can be collected by the microscope objective 13, then focused onto the pinhole 54 via the second reflecting mirror 17, dichroic mirror 12, filter 55, and first achromatic lens 56, and then converted into parallel light by the second achromatic lens 57. The beam splitter 53 can split the fluorescence beam onto the avalanche diode 51 and the spectrometer 52. The avalanche diode 51 can convert the fluorescence beam into fluorescence counting for recording the number of fluorescence photons, and the spectrometer 52 can convert the fluorescence beam into a spectral signal.

[0082] According to an embodiment of the present invention, in the mode of temperature measurement using the All-optical method, the spectral signal can be used as a characteristic signal to determine the temperature change measurement results of the biochemical reaction process.

[0083] According to an embodiment of the present invention, see Figure 5 As shown, when the solid-state spin nanoparticles 27 are irradiated with an excitation beam generated by the pump light module, the microwave module 4 can also generate a microwave field based on the microwave source 42, microwave power amplifier 43, and radiation component 41, radiating it to the solid-state spin nanoparticles 27. In this case, the solid-state spin nanoparticles can absorb the microwave signal. In the mode of temperature measurement using the ODMR method, the absorption frequency of the solid-state spin nanoparticles to the microwave field can be used as a characteristic signal to determine the temperature change measurement results of the biochemical reaction process.

[0084] According to an embodiment of the present invention, see Figure 5 As shown, 6 is the processing module. Processing module 6 can control electronic components such as displacement module 21, fiber optic collimator 16, avalanche diode 51, and spectrometer 52. Processing module 6 can also, for example, record the microwave frequency of the microwave signal generated by microwave source 42.

[0085] Through the above embodiments of the present invention, a submicron scale device for measuring temperature changes during biochemical molecular interactions is provided. The measurement using fluorescence-based nanoparticles and thermally responsive molecules can overcome spatial limitations and has the advantages of simple system structure and operation, high spatial resolution, low sample consumption, good chemical stability, and high sensitivity.

[0086] Figure 6 The flowchart illustrates a method for measuring the temperature of a biochemical reaction based on a solid-state spin sensor, implemented using the ODMR method according to an embodiment of the present invention.

[0087] like Figure 6 As shown, the method includes operations S610 to S670.

[0088] When operating S610, the temperature and humidity of the environment occupied by the sample module are kept stable by the temperature control module. The sample module includes solid spin nanoparticles as temperature probes and test samples for biochemical reactions.

[0089] When operating the S620, an excitation beam is applied to solid spin nanoparticles placed inside the sample module using a pump optical module.

[0090] In operation of S630, the microwave module is used to apply multiple microwave fields of different frequencies to solid spin nanoparticles placed in the sample module.

[0091] By operating the S640 and using an avalanche diode, the fluorescence count of fluorescent photons emitted by solid spin nanoparticles when receiving a microwave field of each frequency is obtained, resulting in multiple fluorescence counts.

[0092] When operating the S650, the microwave frequency of the microwave field is obtained using a microwave source.

[0093] In operating the S660, the processing module controls the simultaneous application of an excitation beam and multiple microwave fields of different frequencies, and records multiple fluorescence counts. When a change in fluorescence count is detected, the target microwave frequency that matches the spin energy level of the solid spin nanoparticle is obtained.

[0094] When operating the S670, the temperature change measurement results of the biochemical reaction process are determined based on the target microwave frequency.

[0095] Figure 7A The diagram illustrates the counting principle of a single fluorescence count according to an embodiment of the present invention.

[0096] According to an embodiment of the present invention, in the mode of temperature measurement using the ODMR method, such as Figure 7A As shown, the processing module 6 can control the pump light module 1 and the microwave module 4 to simultaneously apply an excitation beam and a microwave field of a certain frequency to solid-state spin nanoparticles for a period of time. When an excitation beam is applied to the solid-state spin nanoparticles, they can transition between the ground state and the excited state. During this process, an avalanche diode can be used to detect the fluorescence count of the fluorescent photons emitted by the solid-state spin nanoparticles during this period. After this period ends, the microwave frequency can be changed in the next time period, and the aforementioned process can be repeated to obtain the fluorescence count for the corresponding time period. Using this method, multiple fluorescence counts of fluorescent photons emitted by solid-state spin nanoparticles under different time periods when receiving microwave fields of different frequencies can be obtained.

[0097] According to embodiments of the present invention, when a microwave field is applied to solid-state spin nanoparticles, resonance can occur when the microwave frequency of the microwave field matches the spin energy level of the solid-state spin nanoparticles. When resonance occurs, the fluorescence count of the solid-state spin nanoparticles can change; for example, the count of a spin sensor using an NV color center will decrease. The microwave frequency of the microwave signal at the time of resonance can be used as the resonance frequency. Based on this principle, the microwave frequency at which the fluorescence count changes can be obtained from multiple fluorescence counts and used as the target microwave frequency matching the spin energy level of the solid-state spin nanoparticles. Specifically, in this process, the corresponding microwave frequency can be determined based on the microwave source. By placing the solid-state spin nanoparticles in an environment with changing temperature, keeping the excitation beam signal constant, and simultaneously scanning the microwave frequency of the microwave signal at the time of changing fluorescence counts, the temperature change during the biochemical reaction process can be measured based on the change in the resonance frequency.

[0098] Figure 7B The diagram illustrates a method for determining the resonant frequency based on microwave frequency and fluorescence count according to an embodiment of the present invention.

[0099] Figure 7C The diagram illustrates a method for determining temperature changes based on resonant frequency according to an embodiment of the present invention.

[0100] like Figure 7B As shown, D(T) characterizes the resonance frequency. When solid spin nanoparticles are exposed to an environment where temperature changes are caused by biochemical reactions, the resonance frequency D(T) will change. By detecting the change in resonance frequency, the temperature change can be measured. Figure 7C As shown, the temperature change curve can be determined based on the resonant frequency change curve, thereby determining the temperature change measurement results of the relevant biochemical reaction process.

[0101] According to embodiments of the present invention, the characteristic signal resonance frequency D(T) can also be described using total fluorescence count, four-point method, etc., and is not limited thereto. For example, the resonance frequency D(T) can be determined based on the fluorescence count of the fluorescence beam emitted by the solid spin nanoparticle.

[0102] Figure 8 The flowchart illustrates a method for measuring the temperature of a biochemical reaction based on a solid-state spin sensor, implemented using an all-optical method according to an embodiment of the present invention.

[0103] like Figure 8 As shown, the method includes operations S810 to S840.

[0104] When operating the S810, the temperature and humidity of the environment occupied by the sample module are kept stable by the temperature control module. The sample module includes solid spin nanoparticles as temperature probes and test samples for biochemical reactions.

[0105] In operating the S820, an excitation beam is applied to solid spin nanoparticles placed within the sample module using a pump optical module.

[0106] Using the S830, a spectrometer was used to obtain spectral information of fluorescence emitted by solid-state spin nanoparticles.

[0107] When operating the S840, the processing module determines the temperature change measurement results of the biochemical reaction process based on spectral information.

[0108] According to embodiments of the present invention, in the mode of temperature measurement using the All-optical method, an excitation beam can be applied to solid spin nanoparticles. Upon receiving the excitation beam, the solid spin nanoparticles can transition between the ground state and the excited state. The spontaneous emission fluorescence spectrum of the solid spin nanoparticles from the excited state to the ground state changes with temperature. Based on the changes in spectral peak position, peak area, peak width, and other characteristics, temperature changes in related biochemical reaction processes can be measured.

[0109] Figure 9 The schematic diagram illustrates the principle of temperature measurement using the All-optical method according to an embodiment of the present invention.

[0110] like Figure 9 The diagram illustrates the emission spectra of the same solid-state spin nanoparticle excited at different temperatures using the same wavelength excitation beam. Taking the spin sensor with the NV color center as an example, 910 shows the emission spectrum of NV- when the spin sensor is excited with a 594 nm excitation beam at 30°C, 920 shows the emission spectrum of NV- when the spin sensor is excited with a 594 nm excitation beam at 37°C, and the magnified portion 930 can be seen as a comparison of the zero-phonon line portion at 637 nm of emission spectra 910 and 920. This can be used to determine the fluorescence intensity changes at 637 nm corresponding to different emission spectra.

[0111] Figure 10 An example diagram illustrating the temperature response coefficient of a spectral method according to an embodiment of the present invention is shown.

[0112] According to an embodiment of the present invention, by defining the zero-phonon line area after subtracting the linear background as the Debye-Waller factor, a linear relationship between the change of the Debye-Waller factor and temperature can be obtained:

[0113] ΔT=Φ*ΔDWF / DWF0 (1)

[0114] like Figure 10 As shown. ΔT can characterize the amount of temperature change, Φ can characterize the response coefficient of the Debye-Waller factor to temperature change, ΔDWF can characterize the amount of change of the Debye-Waller factor, and DWF0 can characterize the Debye-Waller factor at the reference temperature.

[0115] Figure 11A The diagram illustrates the detection of temperature changes during an acid-base neutralization reaction using an all-optical method according to an embodiment of the present invention.

[0116] Figure 11B The illustration schematically shows the detection results of the stability of acid-base neutralization reaction using the All-optical method according to an embodiment of the present invention.

[0117] like Figure 11A and Figure 11B As shown, for example, this embodiment of the invention provides a stability test for acid-base neutralization reactions using an all-optical method to measure temperature in a non-isothermal mode. The specific implementation process may include: pre-placing the acid solution in the reaction chamber, placing the alkali solution at the tip of a microfluidic tube, and waiting for the ambient temperature around the reaction chamber to reach thermal equilibrium. Then, the alkali solution is pushed into the reaction chamber from the microfluidic tube, while simultaneously detecting the temperature change corresponding to the fluorescence spectrum emitted by the solid spin nanoparticles. Figure 11A As shown, the acid-base neutralization reaction temperature changes drastically. This can be observed by comparing the initial temperature ΔT. ini and the temperature ΔT after the reaction fin Changes in the baseline can be used to assess the chemical stability of solid spin nanoparticles as temperature probes. Figure 11B It exhibited stability better than 100 mK after undergoing multiple acid-base neutralization reactions.

[0118] Figure 12 The diagram illustrates the detection of temperature changes during the ATP enzymatic hydrolysis process using an all-optical method according to an embodiment of the present invention.

[0119] like Figure 12 The image shows, for example, a temperature change graph illustrating the kinetics of ATP (adenosine triphosphate) hydrolysis under enzymatic catalysis, measured using an all-optical method in a non-isothermal mode, as provided in an embodiment of the present invention. The specific implementation process may include: pre-placing the catalytic enzyme solution in the reaction chamber and the ATP solution in the tip of the microfluidic tube, waiting for the ambient temperature around the reaction chamber to reach thermal equilibrium. Then, ATP is introduced into the reaction chamber, while simultaneously monitoring the temperature change corresponding to the fluorescence spectrum emitted by the solid spin nanoparticles. Figure 12As shown, ATP undergoes hydrolysis under enzyme catalysis, resulting in heat release and a temperature increase, while the system simultaneously dissipates heat, causing a temperature decrease. The entire process is fitted using a double-E exponential function, and the reaction time τ can be obtained from the fitting results. rise and reaction enthalpy H reac =84*ΔT reac The double-E exponential function is shown in equation (2):

[0120]

[0121] ΔT(t) can characterize the temperature change at time t. ini ΔT can characterize the temperature change relative to the reference ambient temperature before the reaction begins. reac It can characterize the temperature change relative to the reference ambient temperature corresponding to the enthalpy of reaction, ΔT fin τ can characterize the temperature change relative to the reference ambient temperature after thermal equilibrium. rise It can characterize reaction time, τ decl It can characterize the heat dissipation time, and t0 can characterize the start time of a biochemical reaction.

[0122] According to an embodiment of the present invention, when it is necessary to perform a biochemical reaction temperature measurement method based on a solid-state spin sensor, the following operations can be performed: S1. Preparation of the reaction chamber: Design the reaction chamber and fix the solid spin nanoparticles inside the reaction chamber. S2. Pre-placement of the reaction solution: Pre-place droplets of reaction solution A in the reaction chamber to immerse the solid spin nanoparticles, and then pre-place reaction solution B in the microfluidic tube. S3. Stabilization of temperature: According to the set parameters of the temperature control module, wait for the temperature near the solid spin nanoparticles to reach equilibrium. S4. Addition of reaction solution B: After temperature equilibrium is reached, push droplets of reaction solution B into the reaction chamber, while continuously detecting the characteristic signals reflected by the solid spin nanoparticles. S5. Data processing: Process the characteristic signals to obtain the time-domain change results of the temperature, thereby obtaining relevant information on the enthalpy of reaction of reaction solutions A and B and the reaction rate.

[0123] The above embodiments of the present invention provide a method for measuring the temperature of biochemical reactions based on solid-state spin sensors. Utilizing the rapid response of solid-state spin sensors to temperature changes, it enables temperature measurement of submicron-scale biomolecular interactions, characterized by good chemical stability, high temperature sensitivity, and low sample consumption. For example, based on the ODMR method, when monitoring temperature changes in biochemical reactions using changes in the absorption frequency of microwave fields by solid-state spin nanoparticles sensitive to temperature changes, it exhibits high robustness to error sources from biological tissues such as scattering, absorption, and background fluorescence, and also possesses high sensitivity. The all-optical temperature measurement method based on spectral information eliminates the need for microwave manipulation, simplifying operation. This device is compatible with both temperature measurement methods, has a simple optical and electronic structure, and boasts low operating costs. The above embodiments of the present invention, combined with wide-field technology, can also be extended to high-throughput applications.

[0124] It should be noted that directional terms mentioned in the embodiments, such as "up," "down," "front," "back," "left," and "right," are only for reference to the directions in the accompanying drawings and are not intended to limit the scope of protection of the present invention. Throughout the drawings, the same elements are represented by the same or similar reference numerals. Conventional structures or constructions will be omitted where they may cause confusion in understanding the present invention. Furthermore, the shapes and sizes of the components in the drawings do not reflect actual size and proportion, but only illustrate the content of the embodiments of the present invention. Moreover, the word "comprising" does not exclude the presence of elements or steps not listed in the claims. The word "a" or "an" preceding an element does not exclude the presence of a plurality of such elements.

[0125] Similarly, it should be understood that, in order to simplify the invention and aid in understanding one or more of the various aspects of the invention, in the above description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof. However, this inventive approach should not be construed as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as reflected in the following claims, inventive aspects lie in fewer than all features of a single embodiment of the foregoing invention. Therefore, the claims following the detailed description are hereby expressly incorporated into this detailed description, wherein each claim itself is a separate embodiment of the invention.

[0126] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A biochemical reaction temperature measurement device based on a solid-state spin sensor, comprising: The sample module is pre-loaded with a sample to be tested and solid spin nanoparticles as a temperature probe. The sample module is used to complete the biochemical reaction of the sample to be tested, and the solid spin nanoparticles are used to measure the temperature change during the biochemical reaction. The sample to be tested includes various reaction liquids or reaction samples capable of carrying out biochemical reactions. Using the solid spin nanoparticles as a temperature probe, the spatial resolution is at the submicron scale, and combined with the sample to be tested, the measurement volume is on the order of femtoliters. A temperature control module is used to maintain a stable ambient temperature and humidity in the area occupied by the sample module. A pump light module is used to generate an excitation beam and focus the excitation beam onto the solid spin nanoparticles so that the target solid spin nanoparticles within the beam range of the excitation beam emit fluorescence. A microwave module for generating a microwave field and radiating the microwave field to the solid spin nanoparticles; A collection module is used to collect a fluorescence beam corresponding to the fluorescence, and to process the fluorescence beam to obtain a fluorescence signal; The processing module is used to receive the fluorescence signal, convert the fluorescence signal into a feature signal, and determine the temperature change measurement result of the biochemical reaction process based on the feature signal. The sample to be tested includes a first sample and a second sample, and the sample module includes: A microdroplet controller for generating a predetermined volume of the first sample; The reaction chamber, consisting of an organic glass ring and a double-layered glass vacuum insulation layer, is used to load the second sample and the solid spin nanoparticles, and to provide reaction space for the biochemical reaction process of the first sample and the second sample, wherein the solid spin nanoparticles are fixed on a glass substrate above the double-layered glass vacuum insulation layer. A microfluidic tube is used to introduce the first sample generated by the microdroplet controller into the reaction chamber; The displacement module is used to carry the reaction chamber and control the solid spin nanoparticles loaded in the reaction chamber to lock at the focusing point of the microscope objective light spot; The temperature control module includes: The outer temperature control submodule is configured to surround the microscope objective and the sample module, and is used to provide a stable ambient temperature for the microscope objective and the sample module; The inner temperature control submodule is configured to surround the sample module and is used to provide a stable ambient temperature and humidity for the solid spin nanoparticles. The inner temperature control submodule includes: A surrounding heating plate is configured to enclose the reaction chamber in conjunction with the displacement module, for providing a stable ambient temperature for the solid spin nanoparticles; A water tank is located around the heating plate to provide stable environmental humidity for the solid spin nanoparticles. A reference thermometer is configured to be connected to the reaction chamber for measuring the ambient temperature of the reaction chamber. A temperature controller is configured to be connected to the reference thermometer and to control the power of the surrounding heating plate based on the ambient temperature measured by the reference thermometer.

2. The apparatus according to claim 1, wherein, The solid-state spin nanoparticles include at least one of the following: nanoparticles containing a temperature-sensitive spin sensor, and a composite probe combining a temperature-responsive material and the spin sensor.

3. The apparatus according to claim 1, wherein, The microwave module includes: A microwave source used to generate a microwave field with modulated spin states; A microwave power amplifier for enhancing the power of the microwave field; A radiation component for radiating the microwave field to the solid spin nanoparticles.

4. The apparatus according to claim 1, wherein, The collection module includes: A filter is used to filter the fluorescent beam. An achromatic lens is used to focus the fluorescent beam onto the pinhole; The pinhole is used for spatial filtering of the fluorescent beam; Beam splitters are used to split the filtered and spatially filtered fluorescence beams and send them to the spectrometer and avalanche diode. The avalanche diode is used to convert the fluorescent beam into a fluorescence count for recording the number of fluorescent photons; The spectrometer is used to convert the fluorescent beam into a spectral signal.

5. The apparatus according to claim 2, wherein, The pump optical module includes: A light source is used to generate an excitation beam with a wavelength corresponding to the spin sensor. A modulator for modulating the intensity of the excitation beam; Fiber collimator is used to project a modulated excitation beam onto a dichroic mirror; The dichroic mirror is used to send the excitation beam to the microscope objective and the fluorescence beam to the collection module. The microscope objective is used to focus the excitation beam onto the solid spin nanoparticles and to collect the fluorescence beam.

6. A method for measuring the temperature of a biochemical reaction based on a solid-state spin sensor, implemented using the apparatus according to any one of claims 1-5, the method comprising: The temperature and humidity of the environment occupied by the sample module are kept stable by a temperature control module. The sample module includes solid spin nanoparticles as temperature probes and test samples for biochemical reactions. An excitation beam is applied to solid spin nanoparticles placed within the sample module using a pump optical module. Using a microwave module, multiple microwave fields of different frequencies are applied to solid spin nanoparticles placed inside the sample module. Using an avalanche diode, the fluorescence count of fluorescent photons emitted by the solid spin nanoparticles when receiving a microwave field of each frequency is obtained, resulting in multiple fluorescence counts. The microwave frequency of the microwave field is obtained using a microwave source. Using a processing module, the excitation beam and the multiple microwave fields of different frequencies are applied simultaneously, and the multiple fluorescence counts are recorded. When a change in the fluorescence count is detected, the target microwave frequency that matches the spin energy level of the solid spin nanoparticle is obtained. The temperature change measurement results of the biochemical reaction process are determined based on the target microwave frequency.

7. A method for measuring the temperature of a biochemical reaction based on a solid-state spin sensor, implemented using the apparatus according to any one of claims 1-5, the method comprising: The temperature and humidity of the environment occupied by the sample module are kept stable by a temperature control module. The sample module includes solid spin nanoparticles as temperature probes and test samples for biochemical reactions. An excitation beam is applied to solid spin nanoparticles placed within the sample module using a pump optical module. The spectral information of the fluorescence emitted by the solid spin nanoparticles was obtained using a spectrometer. Using the processing module, the temperature change measurement results of the biochemical reaction process are determined based on the spectral information.

8. A method for measuring the temperature of a biochemical reaction based on a solid-state spin sensor, implemented using the apparatus according to any one of claims 1-5, the method comprising: Temperature changes during a biochemical reaction in a reaction chamber are measured using solid-state spin nanoparticles. The reaction chamber is located in a sample module, which further includes a microdroplet controller for generating a first sample of a predetermined volume. The reaction chamber is used to load the solid-state spin nanoparticles and a second sample, and to provide reaction space for the biochemical reaction between the first and second samples. The sample module also includes a microfluidic tube for introducing the first sample generated by the microdroplet controller into the reaction chamber, a module for supporting the reaction chamber, and a displacement module for controlling the solid-state spin nanoparticles loaded in the reaction chamber to lock at the focusing point of a microscope objective. A photocooling material placed in the reaction chamber is irradiated with cooling light, causing the photocooling material to absorb the heat released during the biochemical reaction process, thereby maintaining the thermal equilibrium temperature of the reaction chamber consistent with the ambient temperature; and The controller of the photocooling light adjusts the power of the photocooling light based on the temperature feedback from the solid spin nanoparticles. Based on the power change of the photocooling light and the calibration of the absorption power of the photocooling material, the heat release of the biochemical reaction occurring in the reaction chamber is determined. Based on the heat release, the temperature change measurement result of the biochemical reaction is obtained.

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